There are currently no studies based on uniformly selected populations of patients with poly JIA that address comprehensively the mechanisms outlined above. This application proposes a series of studies which will capitalize on the unique opportunity to use samples from the TREAT (The Trial of Aggressive Therapy) clinical trial. TREAT will test whether aggressive early therapy with ETN, prednisolone and MTX will induce an inactive disease (ID) state in patients with poly-JIA more frequently than conventional therapy (MTX alone). Using an appropriately designed and statistically powered approach, this project will identify immune pathways characteristic of treatment responders and non-responders and identify the pathogenic relevance of the cross- talk between various populations of immune cells. This knowledge should also lead to strategies for the rescue of non-responders by adjustments of the therapeutic regimens. These studies will also identify whether mechanisms of immune tolerance/suppression become less efficient in patients who do not maintain ID, or are impaired in patients who never achieve ID. By linking clinical intervention with mechanistic studies, these studies will also identify useful biomarkers of disease activity and treatment efficacy. The central hypothesis for this project is that effective therapies with methotrexate (MTX) alone or in combination with Etanercept (ETN) and prednisolone induce inactive disease (ID) in polyarticular juvenile idiopathic arthritis (poly JIA) by affecting multiple interdependent immune pathways, including T cell regulation (Treg), lineage commitment (Th-1-3, 17) for T effector (Teff) and APC function. It is the interaction among multiple pathways, rather than a single mechanism, which leads to immunological down-regulation and clinical improvement. Specific Aim I: To identify Treg mechanisms which are modified by the treatment; Specific Aim II: To identify treatment-induced functional changes in lineage commitment (TH-1-3, 17) for Teff; Specific Aim III: To identify treatment-induced functional changes in APC; Specific Aim IV: To identify mechanisms of T-APC cross-talk functionally relevant to immune tolerance; Specific Aim V: To identify biomarkers of disease activity and treatment response; Specific Aim VI: To assess the correlation of the findings from the previous aims with clinical outcomes. PUBLIC HEALTH RELEVANCE: Project Narrative (3 sentences Max) The work proposed here provides a fundamental mechanistic-translational outlet to the TREAT clinical trial, connecting molecular immunological mechanisms with clinical outcomes, and is anticipated to contribute to significant advancement in the area of pediatric rheumatology by identifying in detail the effects of state of art therapy on various components of the immune system. This project has the potential to provide valuable information regarding the immunology of human immune tolerance and biomarkers of JIA and/or treatment response. If certain biomarkers are unable to detect differences in this setting then they are even less likely to be helpful in the clinical setting, thus these studies will lay the framework for understanding which tests are and are not worthwhile evaluating in the future for their performance in clinical settings.